Limits...
An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A.

Matoba Y, Miyasako M, Matsuo K, Oda K, Noda M, Higashikawa F, Kumagai T, Sugiyama M - FEBS Open Bio (2014)

Bottom Line: Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit.At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures.A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.

ABSTRACT
A plant-derived Enterococcus mundtii 15-1A, that has been previously isolated from Brassica rapa L. subsp. nipposinica (L.H. Bailey) Hanelt var. linearifolia by our group, possesses two kinds of l-lactate dehydrogenase (l-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial l-LDHs, the structure without the ligands is different from that of any other previously determined l-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the α-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.

No MeSH data available.


Related in: MedlinePlus

Superposition of subunit structures of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. Subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are colored red, yellow, and darkgreen, respectively. Subunits of the Bf. longuml-LDH in the active and inactive states [9] are colored blue and cyan, respectively. In a, subunit A in the active state of the Ec. mundtii LDH-2 are compared with the subunits of the Bf. longuml-LDH in the active and inactive states. In b, subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are superimposed. Superpositions were calculated using all Cα atoms (a) or Cα atoms in the catalytic domain (b). NADH molecule bound to subunit A in the active state of the Ec. mundtii LDH-2 is shown in the stick model colored orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4219987&req=5

f0025: Superposition of subunit structures of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. Subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are colored red, yellow, and darkgreen, respectively. Subunits of the Bf. longuml-LDH in the active and inactive states [9] are colored blue and cyan, respectively. In a, subunit A in the active state of the Ec. mundtii LDH-2 are compared with the subunits of the Bf. longuml-LDH in the active and inactive states. In b, subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are superimposed. Superpositions were calculated using all Cα atoms (a) or Cα atoms in the catalytic domain (b). NADH molecule bound to subunit A in the active state of the Ec. mundtii LDH-2 is shown in the stick model colored orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: Four subunits in the ligands-bound tetramer of the Ec. mundtii LDH-2 are similar to one another. They can be superimposed with root mean square (rms) deviations in a range between 0.20 and 0.48 Å for the 312 Cα atoms. When compared with the active state of the Bf. longuml-LDH, the residues Val100–Leu112 and Val213–Arg220a of the Ec. mundtii LDH-2 are structurally different (Fig. 5a). The residues Val100–Leu112 protrude away from the main body of the subunit, as found in the inactive state of the Bf. longuml-LDH. Since this region is one side of the pyruvate-binding pocket, the binding of pyruvate to the active site may induce the structural change of this region. On the other hand, the residues Val213–Arg220a in the Ec. mundtii LDH-2 are structurally different from the corresponding residues in the Bf. longuml-LDH because former enzyme lacks two residues on the amino acid sequence (Figs. 1 and 4). However, without these residues, each subunit of the ligands-bound Ec. mundtii LDH-2 is structurally similar to that of the Bf. longuml-LDH in the active state with rms deviations in a range between 1.1 and 1.2 Å. It should be noted that an α-helix (α1/2G in Fig. 4) consisting of the residues Glu222–Lys243 is bent around Asn234, as found in the active state of the Bf. longuml-LDH.


An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A.

Matoba Y, Miyasako M, Matsuo K, Oda K, Noda M, Higashikawa F, Kumagai T, Sugiyama M - FEBS Open Bio (2014)

Superposition of subunit structures of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. Subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are colored red, yellow, and darkgreen, respectively. Subunits of the Bf. longuml-LDH in the active and inactive states [9] are colored blue and cyan, respectively. In a, subunit A in the active state of the Ec. mundtii LDH-2 are compared with the subunits of the Bf. longuml-LDH in the active and inactive states. In b, subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are superimposed. Superpositions were calculated using all Cα atoms (a) or Cα atoms in the catalytic domain (b). NADH molecule bound to subunit A in the active state of the Ec. mundtii LDH-2 is shown in the stick model colored orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219987&req=5

f0025: Superposition of subunit structures of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. Subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are colored red, yellow, and darkgreen, respectively. Subunits of the Bf. longuml-LDH in the active and inactive states [9] are colored blue and cyan, respectively. In a, subunit A in the active state of the Ec. mundtii LDH-2 are compared with the subunits of the Bf. longuml-LDH in the active and inactive states. In b, subunit A in the active state and subunits A and B in the inactive state of the Ec. mundtii LDH-2 are superimposed. Superpositions were calculated using all Cα atoms (a) or Cα atoms in the catalytic domain (b). NADH molecule bound to subunit A in the active state of the Ec. mundtii LDH-2 is shown in the stick model colored orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: Four subunits in the ligands-bound tetramer of the Ec. mundtii LDH-2 are similar to one another. They can be superimposed with root mean square (rms) deviations in a range between 0.20 and 0.48 Å for the 312 Cα atoms. When compared with the active state of the Bf. longuml-LDH, the residues Val100–Leu112 and Val213–Arg220a of the Ec. mundtii LDH-2 are structurally different (Fig. 5a). The residues Val100–Leu112 protrude away from the main body of the subunit, as found in the inactive state of the Bf. longuml-LDH. Since this region is one side of the pyruvate-binding pocket, the binding of pyruvate to the active site may induce the structural change of this region. On the other hand, the residues Val213–Arg220a in the Ec. mundtii LDH-2 are structurally different from the corresponding residues in the Bf. longuml-LDH because former enzyme lacks two residues on the amino acid sequence (Figs. 1 and 4). However, without these residues, each subunit of the ligands-bound Ec. mundtii LDH-2 is structurally similar to that of the Bf. longuml-LDH in the active state with rms deviations in a range between 1.1 and 1.2 Å. It should be noted that an α-helix (α1/2G in Fig. 4) consisting of the residues Glu222–Lys243 is bent around Asn234, as found in the active state of the Bf. longuml-LDH.

Bottom Line: Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit.At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures.A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.

ABSTRACT
A plant-derived Enterococcus mundtii 15-1A, that has been previously isolated from Brassica rapa L. subsp. nipposinica (L.H. Bailey) Hanelt var. linearifolia by our group, possesses two kinds of l-lactate dehydrogenase (l-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial l-LDHs, the structure without the ligands is different from that of any other previously determined l-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the α-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.

No MeSH data available.


Related in: MedlinePlus